1. Field of the Invention
The present invention relates to a thrombolytic composition containing tissue type plasminogen activator (t-PA) or a derivative thereof as a major active component.
2. Description of the Prior Art
t-PA has become the object of much attention as a novel plasminogen activator for pharmaceutical use, because, unlike conventionally known urokinase, t-PA has strong affinity to fibrin and high thrombolytic activity and, in particular, single-chain t-PA causes fewer side effects. Recently, it has become possible to use t-PA as a thrombolytic agent practically in medicine due to the development of procedures for producing t-PA in large quantities using cell culture techniques as well as gene recombination techniques.
However, t-PA, a protein having an isoelectric point in the range of 6-8, is extremely insoluble at pHs near the isoelectric point so that it became necessary to solve the problem with regard to the solubility of t-PA in order to formulate t-PA as a medicament. For this purpose, various investigations regarding the t-PA preparation have been conducted to improve the solubility of t-PA, and thus a good deal of information has been provided.
In general, known methods for improving the solubility of proteins include (1) dissolving the protein at an acidic or alkaline pH apart from the isoelectric point of the protein, (2) dissolving the protein at a pH near the isoelectric point of the protein in a solution with an increased salt concentration or ionic strength, and (3) dissolving the protein with the aid of a dissolution auxiliary agent specific to the protein.
Known methods to improve the solubility and stability of t-PA in preparing pharmaceutical t-PA formulations are characterized in that (1) acidic pHs apart from the isoelectric point of t-PA are used (for example, Japanese Patent Laid-open No. 26234/1987 and Japanese Patent Laid-Open No. 36332/1987), (2) the protein is diluted in a solution solely with a t-PA stabilizing agent, in which none of particular dissolution auxiliary agent is added and a salt in a relatively high concentration is used (for example, Japanese Patent Laid-open No. 292729/1987) or (3) a dissolution auxiliary agent such as a basic amino acid (e.g., arginine) is added (for example, Japanese Patent Laid-open No. 81326/1987, Japanese Patent Laid-open 120321/1987, and Japanese Patent Laid-open 164632/1987).
The above-mentioned pharmaceutical methods for the preparation of t-PA improve the solubility of t-PA in different ways but have undesirable disadvantages from a pharmaceutical point of view.
Specifically, (1) it is desirable to prepare pharmaceuticals for injection near the pH of blood (pH 7.4) and an acid solution may cause hemolysis so that a preparation at a pH near neutral is more preferable than that at an acidic pH; (2) a solution with a high salt concentration is not preferable for injection because of its high osmotic pressure; furthermore, in a preparation in which large quantities of salt are added in advance to prepare a lyophilized preparation, the osmotic pressure is increased extremely high when later diluted in physiological saline, which is generally used for dilution; furthermore, considering the use of t-PA as a medicament, it is not desirable to administer sodium ion in large doses; and (3) compounds known as auxiliary agents for the dissolution of t-PA today have the following disadvantages: i) when used alone, none of the agents can dissolve t-PA satisfactorily so that it is necessary to increase the salt concentration to some extent, and ii) when a lyophilized preparation is reconstituted and then diluted exceeding a certain dilution extent with a low salt diluent, the auxiliary agent for dissolution is also diluted at the same time, and the dissolution effect is thus lessened, resulting in precipitation of t-PA in some cases. Consequently, there arises such an inconvenience that the t-PA preparation is well dissolved in physiological saline, which is an infusion for injection with a high salt concentration, but hardly dissolved in a 5% glucose solution which is also generally used as an infusion for injection with a low salt concentration. t-PA is administered mostly in cases of an urgent need and, in such cases, alternative is usually limited for an infusion to reconstitute a lyophilized t-PA preparation or to dilute it; for this reason, it has been desired to develop a pharmaceutical preparation suitable for use in injection, which is easy to dissolve both in physiological saline and in a 5% glucose solution over a broad range of t-PA concentrations while providing an appropriate salt concentration and osmotic pressure. Further, the term "salt" in a salt concentration as used in this specification means mainly chloride; however, it also includes other salts such as amino acids or other compounds in the form of salts which are clinically acceptable.